Self forming, thermodynamically stable liposomes and their applications

ABSTRACT

A lipid composition forms liposomes spontaneously upon mixing with an aqueous solution. The lipid composition includes diacylglycerol-PEG compounds. Such lipid compositions are useful for forming liposomes, which in turn are useful for a variety of purposes, including the delivery of therapeutic agents.

FIELD OF THE INVENTION

The present invention relates to liposomes. More particularly, thepresent invention relates to liposomes which form spontaneously uponmixing of lipids and an aqueous solution, and applications thereof.

BACKGROUND OF THE INVENTION

Liposomes are self-closed colloidal particles in which membranescomposed of one or more lipid bilayer(s) encapsulate a fraction of theaqueous solution in which they are suspended. The surfaces of bilayersare hydrophilic while the interior of bilayers, which containhydrocarbon chains, are hydrophobic. Because of the differentmicroenvironments in their structure, liposomes can encapsulatehydrophilic molecules, bind molecules on the bilayer surfaces ordissolve hydrophobic molecules into the middle of the bilayer. Theirability to incorporate many types of molecules has resulted inapplications for drug delivery, diagnostics, cosmetics, cosmeceuticalsand nutraceuticals.

Liposomes are manufactured by several different methods. Typically, theprocess begins when a lipid or combination of lipids are dissolved in anorganic solvent. Upon removal of organic solvents and hydration, largemultilamellar vesicles (MLVs) are formed. For some applications, smallunilamellar vesicles (SUVs) may be desired. SUVs can be produced fromMLVs by several techniques including sonication, extrusion throughmembranes with well-defined pores, French press extrusion andhomogenization.

Problems associated with liposomes include colloidal instability,difficulty in scale-up sterilization, and variability between batches inmanufacturing. Liposome preparation and manufacturing typically involvesremoval of organic solvents followed by extrusion or homogenization.These processes may expose liposomal components to extreme conditionssuch as elevated pressures, elevated temperatures and high shearconditions which can degrade lipids and other molecules incorporatedinto the liposomes.

Liposome preparations are often characterized by very heterogeneousdistributions of sizes and number of bilayers. Conditions optimized on asmall scale normally do not scale up well and preparation of large-scalebatches is cumbersome and labor intensive.

Another issue associated with liposomes for medical uses issterilization. Among heat sterilization, ethanol oxide exposure, gammairradiation and sterile filtration, only the last technique is suitablefor liposomes and then only for liposomes smaller than about 100nanometers (nm). Filtration of liposomes poses many difficulties.

Another problem for liposome applications is colloidal stability.Liposomes in suspension can aggregate and fuse upon storage, heating andaddition of various additives. Because of these stability problems,liposomes are often lyophilized. Lyophilization is costly and timeconsuming. Upon reconstitution, size distributions often increase andencapsulated materials may leak out from the liposomes.

It is therefore desirable to develop new methods and materials whichaddress these problems with current liposome formulations.

BRIEF DESCRIPTION OF THE INVENTION

A liposome suspension forms spontaneously upon adding a lipidcomposition to an aqueous solution. The lipid composition comprises asingle lipid or a mixture of lipids that have appropriate packingparameters, that includes polyethyleneglycol, and that has a meltingtemperature which allows it to be in liquid form when mixed with theaqueous solution. Such liposome suspensions are useful for a variety ofpurposes, including the delivery of therapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent invention and, together with the detailed description, serve toexplain the principles and implementations of the invention.

In the drawings:

FIG. 1 is a diagram depicting the cross-section of a liposome made oflipid molecules.

FIG. 2 is a space-filling diagram of a lipid molecule having a polarhead group and nonpolar hydrocarbon chains.

FIG. 3 is a diagram depicting a cross-section of a micelle made of lipidmolecules.

FIG. 4 is a diagram depicting a cross-section of a structure made oflipid molecules with large tails relative to the head groups.

FIG. 5 is a diagram showing the molecular structure of PEG-12 GlycerylDioleate or Hetoxamate GDO-12.

DETAILED DESCRIPTION

Embodiments of the present invention are described herein in the contextof a self-forming, thermodynamically stable liposomes and theirapplications. Those of ordinary skill in the art will realize that thefollowing detailed description of the present invention is illustrativeonly and is not intended to be in any way limiting. Other embodiments ofthe present invention will readily suggest themselves to such skilledpersons having the benefit of this disclosure. Reference will now bemade in detail to implementations of the present invention asillustrated in the accompanying drawings. The same reference indicatorswill be used throughout the drawings and the following detaileddescription to refer to the same or like parts.

In the interest of clarity, not all of the routine features of theimplementations described herein are shown and described. It will, ofcourse, be appreciated that in the development of any such actualimplementation, numerous implementation-specific decisions must be madein order to achieve the developer's specific goals, such as compliancewith application- and business-related constraints, and that thesespecific goals will vary from one implementation to another and from onedeveloper to another. Moreover, it will be appreciated that such adevelopment effort might be complex and time-consuming, but wouldnevertheless be a routine undertaking of engineering for those ofordinary skill in the art having the benefit of this disclosure.

Those of ordinary skill in the art will realize that the followingdescription of the present invention is illustrative only and not in anyway limiting. Other embodiments of the invention will readily suggestthemselves to such skilled persons having the benefit of thisdisclosure.

Most, if not all, known liposome suspensions are not thermodynamicallystable. Instead, the liposomes in known suspensions are kineticallytrapped into higher energy states by the energy used in their formation.Energy may be provided as heat, sonication, extrusion, orhomogenization. Since every high-energy state tries to lower its freeenergy, known liposome formulations experience problems withaggregation, fusion, sedimentation and leakage of liposome associatedmaterial. A thermodynamically stable liposome formulation which couldavoid some of these problems is therefore desirable.

The present invention teaches liposome suspensions which arethermodynamically stable at the temperature of formation. Theformulation of such suspensions is achieved by employing a compositionof lipids having several fundamental properties. First, the lipidcomposition must have packing parameters which allow the formation ofliposomes. Second, as part of the head group, the lipid should includepolyethyleneglycol (PEG) or any polymer of similar properties whichsterically stabilizes the liposomes in suspension. Third, the lipid musthave a melting temperature which allows it to be in liquid form whenmixed with an aqueous solution.

By employing lipid compositions having the desired fundamentalproperties, little or no energy need be added when mixing the lipid andan aqueous solution to form liposomes. When mixed with water, the lipidmolecules disperse and self assemble as the system settles into itsnatural low free energy state. Depending on the lipids used, the lowestfree energy state may include small unilamellar vesicle (SUV) liposomes,multilamellar vesicle (MLV) liposomes, or a combination of SUVs andMLVs.

Lipid compositions suitable for use in the invention may includecompositions comprising only a single type of lipid molecule as well ascompositions made up of more than one lipid. As will be appreciated bythose skilled in the art, both types of compositions may be quantifiedaccording to cited fundamental properties.

One required fundamental property is the ability to form liposomes byvirtue of having the proper packing parameters. Packing parameters arerelative measures of a given lipid composition, and depend on factorssuch as size relationships between lipid head groups and lipidhydrocarbon chains, charge, and the presence of stabilizers such ascholesterol. (Israelachvili, D D Lasic, Liposomes: From Physics toApplications, Elsevier, pp 51, 1993).)

To form a lipid bilayer, lipid head groups and hydrocarbon chains mustorganize themselves so that the radius of curvature results in aliposome (see FIG. 1). If the hydrocarbon chains are too small relativeto the head group, the radius of curvature will be too large andmicelles will be produced (see FIG. 3). If the hydrocarbon chains aretoo large relative to the head groups, the radius of curvature will beof the opposite sign and liposomes cannot form (see FIG. 4).

FIG. 1 is a diagram depicting the cross-section of a liposome made oflipid molecules. Liposome 10 comprises a lipid bilayer, made of lipidmolecules (e.g., 12, 14, 16, 18), enclosing an aqueous space 20.

FIG. 2 is a space-filling diagram of a lipid molecule having a polarhead group and nonpolar hydrocarbon chains. Lipid molecule 22 iscomprised of a hydrophilic group 24 and a hydrophobic tail 26.Hydrophobic tail 26 may comprise two hydrocarbon chains 32, 34. Whileits chemical bonds allow the lipid molecule to be flexible, the headgroup generally fills an area of diameter 28 while the tail fills anarea of diameter 30. Because lipid molecules must be organized in abilayer to form a liposome, the ratio of the head group diameter to thetail diameter can be neither too large nor too small if liposomeformation is to occur.

FIG. 3 is a diagram depicting a cross-section of a micelle made of lipidmolecules. Micelle 36 is composed of lipid molecules (e.g., 38, 40).Because the tail groups of the lipid molecules have small diametersrelative to the head groups, the lipid molecules organize with a smallradius of curvature, and a bilayer cannot form.

FIG. 4 is a diagram depicting a cross-section of a structure made oflipid molecules with large tails relative to the head groups. In FIG. 4,it can be seen that structure 42 forms when lipids (e.g., 44) havinglarge tails relative to the head groups are mixed in aqueous solution.Again, the size ratio between head groups and tails makes bilayerformation impossible.

While FIGS. 1, 3 and 4 have illustrated the basic principle of packingparameters using a single type of lipid molecule, it will be appreciatedthat the same principle applies to mixtures of lipids. For example, alipid which has hydrocarbon chains too small to form liposomes as asingle species can be mixed with cholesterol to result in a compositionwhich has the proper packing parameters. As another example, a lipidwhich by itself has the proper packing parameters may form liposomesincorporating limited amounts of other lipids which, by themselves, donot have proper packing parameters. Both single lipids and mixtures oflipids have packing parameters that may be calculated by known methods.(ref) In general, liposome compositions which allow liposome formationhave packing parameter measurements of P_(a) between about 0.84 and 0.88and P_(v) between about 0.88 and 0.93.

P_(a) is the packing parameter with respect to surface and Pv is packingparameter with respect to volume (DD Lasic, Liposomes: From Physics toApplications, Elsevier, pp 51, 1993). The parameters are derived fromthe equations HC_(a)/T_(a)≡P_(a) and HC_(v)/T_(v)≡P_(v) where HC_(a) isthe hydrocarbon chain area, T_(a) is the total area of the molecule,HC_(v) is the volume of the hydrocarbon chains and T_(v) is the volumeof the whole molecule.

Packing parameters can be calculated for mixtures of lipids, since idealmixing of lipids results in arithmetic average of their individualcharacteristics. For instance HC_(a)/T_(a)≡P_(a) of a binary mixture, inthe case of ideal mixing can be expressed as:<P _(a) >=X ₁ P ₁ +X ₂ P ₂ , X ₁ +X ₂=1

More generally in the case of i lipids composing a given mixture can berepresented by:<P _(a) >=Σ _(i) X _(i) P _(i) and Σ_(i) X _(i)=Iwhere X_(i) is the mole fraction of the lipid in the mixture and P_(i)is the packing parameter with respect to surface of that lipid.

FIG. 5 is a diagram showing the molecular structure of PEG-12 GlycerylDioleate or Hetoxamate GDO-12 which includes a glycerol backbone 46, aPEG chain 48, and a tail group 50 having two hydrocarbon chains.Together, the backbone 46 and the PEG chain 48 comprise the head groupof the molecule. PEG-12 GDO is an preferred lipid molecule for use inthe present invention, in part because its head group has the propersize in relation to its tail group.

Two general factors influence the size of the head group in a lipidmolecule. One is the actual physical size of the head group. Forexample, employing a longer PEG chain would make the head group larger.The other is the charge associated with the head group. For example, ifthe PEG chain was conjugated to the backbone by a phosphodiester bondthe phosphate would impart a charge to the head group, effectivelyincreasing its size. In the present invention, non-phospolipids arepreferred so that the general means of varying head group size is byvarying the length of the PEG chain. However, those skilled in the artwill recognize that the effective size of the head group could be variedin other ways, for example by using a different backbone other thanglycerol or adding a linker such as phosphate between the glycerolbackbone and the PEG chain.

The size of the tail of a lipid is mostly influenced by the length ofhydrocarbon chains and degree of saturation in the lipid chain. Singlechain lipids will generally not form liposomes, though they may beincorporated into liposomes composed of lipids with two chains.Similarly, lipids with one long chain and one short chain may haverelatively small tail sizes. Those skilled in the art will recognizethat such lipids may be used to form liposomes in the present invention,especially if sterols such as cholesterol are used to stabilize thebilayer.

A second fundamental property of lipid compositions suitable for use inthis invention is that the head group of at least one lipid must includea PEG chain. PEG stabilizes the liposomes by creating a steric barrierat the outer surface of the liposomes. Preferably, the PEG chain have amolecular weight between about 300 Daltons and 5000 Daltons, althoughthose skilled in the art will recognize that differing concentrations ofPEG on the liposome surface as well as differing chain lengths may beused to stabilize the liposomes.

A third fundamental lipid property is that the lipid composition musthave a melting temperature which allows the composition to be in liquidform when mixed with an aqueous solution. Generally, this means that thelipid composition should have a phase transition temperature of betweenabout 0° C. and 100° C. As with packing parameters, melting temperaturesmay be determined for mixtures of lipids.

Related to melting point of a lipid is bending elastic modulus.Generally preferred in the present invention are lipids with a bendingelastic modulus that allows the lipid to be sufficiently flexible toform liposomes in an aqueous solution without the need for large energyinputs. If the bending elastic modulus is too great, the lipid will betoo rigid to spontaneously form liposomes in an aqueous solution.Preferably, the bending elastic modulus is between about 0 kt and 15 kt.More preferably, the bending elastic modulus is between about 1 kt and10 kt. The bending elastic modulus is largely determined by thebackbone. Glycerol provides an ideal backbone for the present invention.

Table 1 shows a number of lipids which have been tested as single lipidsfor suitability for the present invention. Lipids were tested at 2weight percent in aqueous solution. Note that GDL means glyceroldilaurate, GDO means glycerol dioleate, GDM means glycerol dimyristate,GDP means glycerol dipalmitate, and GDS means glycerol distearate. Foreach lipid, the number after “PEG” indicates the numbers of C₂H₄Osubunits in the PEG chain. The unsaturated dioleate lipids have similarpacking parameters to the saturated dimyristate lipids. meltingSpontaneous Spontaneous Spontaneous point Liposomes at Liposomes atLiposomes at lipid (° C.) P_(a) P_(v) 20° C. 37° C. 60° C. PEG-23 GDLFluid @ 25 .829 .869 NO NO NO PEG-12 GDO Fluid @ 25 YES YES YES PEG-23GDO Fluid @ 25 NO NO NO PEG-45 GDO 36.3 NO NO NO PEG-12 GDM Fluid @ 25.853 .889 YES YES YES PEG-23 GDM Fluid @ 25 .837 .875 NO NO NO PEG-45GDM 33.2 .823 .863 NO NO NO PEG-23 GDP 31.2 .843 .880 YES YES PEG-45 GDP41.8 .828 .867 NO NO NO PEG-12 GDS 40.0 .869 .901 NO NO YES PEG-23 GDS39.8 .849 .885 NO NO YES PEG-45 GDS 40.8 .830 .870 NO NO NO

The table shows that lipids which possess the required properties willspontaneously form liposomes when mixed in an aqueous solution. Forexample, PEG-12 GDM spontaneously forms liposomes at all temperaturestested since it is a liquid at those temperatures and includes PEG inaddition to having packing parameters within the required ranges.Similarly, PEG-12 GDO, which shares nearly identical properties toPEG-12 GDM, spontaneously forms liposomes at all temperatures tested.

PEG-12 GDS shows one example of the requirement that the lipid be liquidat the temperature of liposome formation. While this lipid has therequired packing parameters as well as including PEG, it does notspontaneously form liposomes until the temperature of liposome formationis high enough for the lipid to be in liquid form.

The GDM series of lipids illustrates the importance of proper packingparameters. While these lipids all include PEG and are in liquid form at60 degrees, only PEG-12 GDM has the proper packing parameters to allowspontaneous liposome formation. The GDS lipid series at 60 degreesillustrates the same point.

Those skilled in the art can practice the present invention by usingknowledge of the required properties to predict and create lipidcompositions which will spontaneously form liposomes. Foe example,certain PEG lipids which form micelles can form liposomes in mixtureswith sterols, such as cholesterol, because of cholesterol's effect onpacking parameters and melting point. For instance, the mixture ofPEG-45 GDS and cholesterol forms liposomes. Similarly, the size of thehead group may be changed to affect packing parameters, for example byvarying the size of the PEG chain or by varying the concentration ofPEG-containing lipids in the lipid composition.

It will be appreciated that, while liposomes form spontaneously at thetemperature of formation, cooling of liposomes after formation resultsin liposomes in trapped kinetic states. To minimize distortions inliposome structure upon such cooling, sterols such as cholesterol may bemixed with the lipid before liposome formation. It has been observedthat cholesterol may be dissolved in PEG-12 GDO at up to about 10percent by weight.

Utility

The present invention is useful in a variety of situations, and providesadvantages over the prior art in several different ways. Problems withliposome preparation, reproducibility, colloidal stability,sterilization, and storage may be reduced by employing the invention.

Since the liposomes of the present invention are self-forming, liposomepreparation entails merely mixing the lipid with an aqueous solution. Ingeneral, liposome formation is scale dependant. It is simple, in thecase of the present invention, to scale up from test batches to largebatches.

Because the liposomes of the present invention exist in the lowestenergy state that the lipid can exist in while in aqueous solution,reproducibility of liposome formation is no problem. A defined lipid,lipid mixture, or lipid/compound mixture will form similar liposomesevery time when mixed with the same aqueous solution. It should benoted, however, that above critical concentrations (around 20% weight tovolume for most lipids) non-liposomal structures will begin to form inaqueous solution.

Aggregation and fusion may occur with liposomes in thermodynamicallytrapped states. Because the liposomes of the present invention are inthe lowest energy state, they do not aggregate and fuse.

Because the liposomes of the present invention are small, they can besterile filtered. Also, the lipids may be heat sterilized prior toliposome formation.

As with any liposome dispersion, these liposomes can be lyophilized inthe presence of appropriate cryoprotectants. Even in liquid form, theliposomes are colloidally stable because they are a thermodynamicallystable system. Also, because of their self-forming nature, the liposomesneed not be stored at all. Instead, the lipid may be stored andconstituted into liposomes as needed.

Applications of the present liposomes include the delivery oftherapeutics and other compounds, use in cosmetics, and use in drugdelivery screening.

Since the liposomes include an aqueous space, a hydrophobic regionwithin the bilayer, and sites for covalent attachment (e.g., on the PEGchain or the backbone), many types of compounds may be encapsulated bythe liposomes. Such compounds include compounds ranging from hydrophilicto hydrophobic, including many insoluble compounds. These liposomes maysubstitute for currently available Cremophor® and Solutol®.

For delivery of intravenous drugs, the drug may be provided in a sealedcontainer co-dissolved or co-mixed along with the lipid. The containermay also contain inert gas to reduce decomposition of the drug andlipid. Prior to administration an aqueous solution is added to thecontainer, thereby forming liposomes with encapsulated drug. Drugssuitable for such a mode of administration include: proteins, peptides,nucleic acids, antineoplastic agents, anti-inflammatories,anti-infectives, gastrointestinal agents, biological and immunologicagents, dermatologic agents, ophthalmic and otic agents, diagnosticaids, nutrients and nutritional agents, hematological agents, endocrineand metabolic agents, cardiovasculars, renal and genitourinary agents,respiratory agents, central nervous system agents.

In one aspect, the invention includes a method of preparing liposomes.The method comprises providing an aqueous solution; providing a lipidsolution, where the solution has a P_(a) between about 0.84 and 0.88, aP_(v) between about 0.88 and 0.93, and where at least one lipid in thesolution includes a polyethyleneglycol (PEG) chain; and combining thelipid solution and the aqueous solution. The PEG chain preferably has amolecular weight between about 300 Daltons and 5000 Daltons. Kineticenergy, such as shaking or vortexing, may be provided to the lipidsolution and the aqueous solution. The lipid solution may comprise asingle lipid. The lipid may comprise dioleolglycerol-PEG-12, eitheralone or as one of the lipids in a mixture. The method may furthercomprise providing an active compound; and combining the active compoundwith the lipid solution and the aqueous solution.

The active compound may be selected from the group comprising proteins,peptides, nucleic acids, antineoplastic agents, anti-inflammatories,anti-infectives, gastrointestinal agents, biological and immunologicagents, dermatologic agents, ophthalmic and otic agents, diagnosticaids, nutrients and nutritional agents, hematological agents, endocrineand metabolic agents, cardiovasculars, renal and genitourinary agents,respiratory agents, central nervous system agents.

In another aspect, the invention includes a liposome suspension. Thesuspension comprises one or more lipids, where the lipids as anaggregate have a P_(a) between about 0.84 and 0.88, a P_(v) betweenabout 0.88 and 0.93 and a melting temperature of between about 0 to 100degrees centigrade; and where at least one lipid includes apolyethyleneglycol (PEG) chain. The PEG chain preferably has a molecularweight between about 300 Daltons and 5000 Daltons. The suspension maycomprise a single lipid. The lipid may comprise dioleolglycerol-PEG-12.The suspension may further comprise an active compound, which may beselect from the group described above.

In another aspect, the invention includes a composition for combiningwith an aqueous solution to form a liposome suspension. The compositioncomprises one or more lipids, where the lipids as an aggregate have aP_(a) between about 0.84 and 0.88, a P_(v) between about 0.88 and 0.93and a melting temperature of between about 0 to 100 degrees centigrade;and where at least one lipid includes a polyethyleneglycol (PEG) chain.The PEG chain preferably has a molecular weight between about 300Daltons and 5000 Daltons. The composition may comprise a single lipid.The composition may comprise dioleolglycerol-PEG-12. The composition mayfurther comprise an active compound selected from the group above. Thecomposition may be provided in a sealed container, where the containeralso contains an inert gas to prevent oxidative degradation.

In another aspect, the invention includes a method of intravenouslyadministering a therapeutic compound. The method comprises providing acomposition including one or more lipids, where the lipids as anaggregate have a P_(a) between about 0.84 and 0.88, a P_(v) betweenabout 0.88 and 0.93 and a melting temperature of between about 0 to 100degrees centigrade; and where at least one lipid includes apolyethyleneglycol (PEG) chain; providing an active compound; providingan aqueous solution; combining the composition, compound and solution toform a liposome suspension; and administering the liposome suspensionintravenously. The method may further comprise providing kinetic energyto the liposome suspension. The method may also include providing thecomposition in a sealed container containing an inert gas. The PEG chainpreferably has a molecular weight between about 300 Daltons and 5000Daltons. The composition may comprise a single lipid. The lipid maycomprise dioleolglycerol-PEG-12. The active compound may be selectedfrom the group above.

In another aspect, the invention includes a method of solubilizing anactive compound. The method comprises providing a composition includingone or more lipids, where the lipids as an aggregate have a P_(a)between about 0.84 and 0.88, a P_(v) between about 0.88 and 0.93 and amelting temperature of between about 0 to 100 degrees centigrade; andwhere at least one lipid includes a polyethyleneglycol (PEG) chain;providing the active compound; providing an aqueous solution; andcombining the active compound, the lipid and the aqueous solution toform a liposome suspension. The method may further comprise providingkinetic energy to the liposome suspension. The method may includeproviding the composition in a sealed container containing an inert gas.The PEG chain preferably has a molecular weight between about 300Daltons and 5000 Daltons. The composition may comprise a single lipid.The lipid may comprise dioleolglycerol-PEG-12. The active compound maybe selected from the group above.

In another aspect, the invention includes a method of orallyadministering a therapeutic compound. The method comprises providing acomposition including one or more lipids, where the lipids as anaggregate have a P_(a) between about 0.84 and 0.88, a P_(v) betweenabout 0.88 and 0.93 and a melting temperature of between about 0 to 100degrees centigrade; and where at least one lipid includes apolyethyleneglycol (PEG) chain; providing an active compound; providingan aqueous solution; combining the composition, compound and solution toform a liposome suspension; and administering the liposome suspensionorally in the form selected from the group comprising a two piece hardgelatin capsule, a soft gelatin capsule, or drops.

The compositions may be administered topically, inter-orally, vaginallyor rectally.

While embodiments and applications of this invention have been shown anddescribed, it would be apparent to those skilled in the art having thebenefit of this disclosure that many more modifications than mentionedabove are possible without departing from the inventive concepts herein.The invention, therefore, is not to be restricted except in the spiritof the appended claims.

EXAMPLES Example 1 Spontaneous Liposomes

Single lipids were tested for their ability to spontaneously formliposomes when mixed with an aqueous solution. PEG-12 GDO was obtainedfrom Global 7 (New Jersey). All other lipids were synthesized for theexperiment. For each experiment, water and lipid were separatelyequilibrated at the appropriate temperature. Two weight percent lipidwas added to the aqueous solution, and the solution was analyzed for thepresence of liposomes by optical light microscopy, cryo TEM, freezefracture TEM, or hot stage microscopy. The results of the experimentsare shown in Table 1 above.

Example 2 Formation of Multilamellar Liposomes with Cholesterol

Cholesterol, 10 Wt. %, was added to PEG-12 Glyceryl Dioleate and heatedto about 60°-70° C., for 15-20 minutes. Water, at room temperature, wasadded to the heated lipid solution. The mixture was left overnight.Examination of the preparation under a optical microscopic withpolarized light at 100 power and 600 power showed mulitlamellarliposomes in the size range of 20 nm to 40 nm. No crystals ofcholesterol were observed.

Example 3 Spontaneous Liposomes for Intravenously AdministeringTherapeutic Compounds

Ingredient Conc. (% wt) Methotrexate 5 PEG-12 GDO 10 DI Water 85

Methotrexate (Sigma) was dissolved in PEG-12 Glyceryl Dioleate andgently mixed for about 5 minutes. The resultant mixture was a clearsolution. Deionized water was slowly added to the solution and gentlymixed. The resultant mixture was a yellow, opaque solution. Thepreparation was examined under a light microscope with polarized lightand showed a suspension of liposomes.

Example 4 Spontaneous Liposomes for Intravenously AdministeredTherapeutic Compound and as a Drug Solubilization Vehicle

Ingredient Conc. (wt %) Cyclosporine A 0.77 PEG-12 GDO 4.30 DI Water95.23Cyclosporin A (Sigma 49H4066) was mixed with PEG-12 Glyceryl Dioleate byvortexing and sonication for 10 minutes. Water was added and gentlymixed. Examination under optical microscope at 600 power showedmulitlamelar liposomes and crystals of cyclosporin A.

Example 5 Spontaneous Liposomes with Active Compounds for Dermatology

Ingredient Conc. (wt) PEG-12 GDO 18 g Betamethasone diproprionate 50 mgCholesterol 100 mg Uniphen-23 ® 1.5 mg Water 80.35 g

Weighed amounts of PEG-12 Glyceryl Dioleate, Betamethasone diproprionateand cholesterol were combined and heated to 50° C. while mixing.Uniphen-23® and water were combined an heated to 50° C. When mixturesreached temperature they were commingled while stirring gently. Mixturewas cooled to room temperature while stirring. Examination by opticalmicroscope at 100× and 600× showed a suspension of multilamellarliposomes.

Example 6 Spontaneous Liposomes with Active Compounds for TopicalAnesthesia

Ingredient Conc. Tetracaine   2 g PEG-12 GDO   20 g Uniphen-23 ®  1.5 gWater 76.5 g

Tetracaine, PEG-12 Glyceryl Dioleate, and Uniphen-23® were mixedtogether and heated to 40° C. while stirring. Water was heated to 40degrees C. and added to the tetracaine solution while stirring gently.Mixture was cooled to room temperature. Examination by electronmicroscope showed LUV's and MLV's.

Example 7 Spontaneous Liposomes for Intravenous and Topical Formulations

Tretinoin (all-trans retinoic acid), 6 mg, was dissolved in 500 ul ofPEG-12 Glyceryl Dioleate. Dissolution was complete. Distilled water, 4.5ml, was added to the mixture and gently mixed. This yielded aconcentration of 1 mg/ml. Examination by optical microscope showedmultilamellar liposomes in the size range of 100 nm to 200 nm. Thissolution can easily be incorporated into a cream, gel or lotion dosageform.

While embodiments and applications of this invention have been shown anddescribed, it would be apparent to those skilled in the art having thebenefit of this disclosure that many more modifications than mentionedabove are possible without departing from the inventive concepts herein.The invention, therefore, is not to be restricted except in the spiritof the appended claims.

1. A method of preparing liposomes, the method comprising: providing anaqueous solution; providing a lipid solution, where the solution has aP_(a) between about 0.84 and 0.88, a P_(v) between about 0.88 and 0.93;and where at least one lipid in the solution includes apolyethyleneglycol (PEG) chain, and combining the lipid solution and theaqueous solution.
 2. The method of claim 1, where the PEG chain has amolecular weight between about 300 Daltons and 5000 Daltons
 3. Themethod of claim 1, further comprising: providing kinetic energy to thelipid solution and the aqueous solution.
 4. The method of claim 1, wherethe lipid solution comprises a single lipid.
 5. The method of claim 1,where the lipid comprises dioleolglycerol-PEG-12.
 6. The method of claim1, further comprising: providing an active compound; and combining theactive compound with the lipid solution and the aqueous solution.
 7. Themethod of claim 6, where the active compound is selected from the groupcomprising proteins, peptides, nucleic acids, antineoplastic agents,anti-inflammatories, anti-infectives, gastrointestinal agents,biological and immunologic agents, dermatologic agents, ophthalmic andolic agents, diagnostic aids, nutrients and nutritional agents,hematological agents, endocrine and metabolic agents, cardiovasculars,renal and genitourinary agents, respiratory agents, central nervoussystem agents.
 8. A liposome suspension, the suspension comprising: oneor more lipids, where the lipids as an aggregate have a P_(a) betweenabout 0.84 and 0.88, a P_(v) between about 0.88 and 0.93 and a meltingtemperature of between about 0 to 100 degrees centigrade; and where atleast one lipid includes a polyethyleneglycol (PEG) chain.
 9. Thesuspension of claim 8, where the PEG chain has a molecular weightbetween about 300 Daltons and 5000 Daltons.
 10. The suspension of claim8, where the suspension comprises a single lipid.
 11. The suspension ofclaim 8, where the lipid comprises dioleolglycerol-PEG-12.
 12. Thesuspension of claim 8, further comprising: an active compound.
 13. Thesuspension of claim 12, where the active compound is selected from thegroup comprising proteins, peptides, nucleic acids, antineoplasticagents, anti-inflammatories, anti-infectives, gastrointestinal agents,biological and immunologic agents, dermatologic agents, ophthalmic andotic agents, diagnostic aids, nutrients and nutritional agents,hematological agents, endocrine and metabolic agents, cardiovasculars,renal and genitourinary agents, respiratory agents, central nervoussystem agents.
 14. A composition for combining with an aqueous solutionto form a liposome suspension, the composition comprising: one or morelipids, where the lipids as an aggregate have a P_(a) between about 0.84and 0.88, a P_(v) between about 0.88 and 0.93 and a melting temperatureof between about 0 to 100 degrees centigrade; and where at least onelipid includes a polyethyleneglycol (PEG) chain.
 15. The composition ofclaim 14, where the PEG chain has a molecular weight between about 300Daltons and 5000 Daltons.
 16. The composition of claim 14, where thecomposition comprises a single lipid.
 17. The composition of claim 14,where the lipid comprises dioleolglycerol-PEG-12.
 18. The composition ofclaim 14, further comprising: an active compound.
 19. The suspension ofclaim 18, where the active compound is selected from the groupcomprising proteins, peptides, nucleic acids, antineoplastic agents,anti-inflammatories, anti-infectives, gastrointestinal agents,biological and immunologic agents, dermatologic agents, ophthalmic andotic agents, diagnostic aids, nutrients and nutritional agents,hematological agents, endocrine and metabolic agents, cardiovascularsrenal and genitourinary agents, respiratory agents, central nervoussystem agents.
 20. The composition of claim 14, where the composition isin a sealed container, said container also containing an inert gas. 21.A method of intravenously administering a therapeutic compound, themethod comprising: providing a composition including one or more lipids,where the lipids as an aggregate have a P_(a) between about 0.84 and0.88, a P_(v) between about 0.88 and 0.93 and a melting temperature ofbetween about 0 to 100 degrees centigrade; and where at least one lipidincludes a polyethyleneglycol (PEG) chain; providing an active compound;providing an aqueous solution; combining the composition, compound andsolution to form a liposome suspension; and administering the liposomesuspension intravenously.
 22. The method of claim 21, furthercomprising: providing kinetic energy to the liposome suspension.
 23. Themethod of claim 21, where said providing a composition includesproviding the composition in a sealed container containing an inert gas.24. The method of claim 21, where the PEG chain has a molecular weightbetween about 300 Daltons and 5000 Daltons.
 25. The method of claim 21,where the composition comprises a single lipid.
 26. The method of claim21, where the lipid comprises dioleolglycerol-PEG-12.
 27. The method ofclaim 21, where the active compound is selected from the groupcomprising proteins, peptides, nucleic acids, antineoplastic agents,anti-inflammatories, anti-infectives, gastrointestinal agents,biological and immunologic agents, dermatologic agents, ophthalmic andotic agents, diagnostic aids, nutrients and nutritional agents,hematological agents, endocrine and metabolic agents, cardiovasculars,renal and genitourinary agents, respiratory agents, central nervoussystem agents.
 28. A method of solubilizing an active compound, themethod comprising: providing a composition including one or more lipids,where the lipids as an aggregate have a P_(a) between about 0.84 and0.88, a P_(v) between about 0.88 and 0.93 and a melting temperature ofbetween about 0 to 100 degrees centigrade; and where at least one lipidincludes a polyethyleneglycol (PEG) chain; providing the activecompound; providing an aqueous solution; and combining the activecompound, the lipid and the aqueous solution to form a liposomesuspension.
 29. The method of claim 28, further comprising: providingkinetic energy to the liposome suspension.
 30. The method of claim 28,where said providing a composition includes providing the composition ina sealed container containing an inert gas.
 31. The method of claim 28,where the PEG chain has a molecular weight between about 300 Daltons and5000 Daltons.
 32. The method of claim 28, where the compositioncomprises a single lipid.
 33. The method of claim 28, where the lipidcomprises dioleolglycerol-PEG-12.
 34. The method of claim 28, where theactive compound is selected from the group comprising proteins,peptides, nucleic acids, antineoplastic agents, anti-inflammatories,anti-infectives, Gastrointestinal agents, biological and immunologicagents, dermatologic agents, ophthalmic and otic agents, diagnosticaids, nutrients and nutritional agents, hematological agents, endocrineand metabolic agents, cardiovasculars, renal and genitourinary agents,respiratory agents, central nervous system agents.
 35. A method oforally administering a therapeutic compound, the method comprising:providing a composition including one or more lipids, where the lipidsas an aggregate have a P_(a) between about 0.84 and 0.88, a P_(v)between about 0.88 and 0.93 and a melting temperature of between about 0to 100 degrees centigrade; and where at least one lipid includes apolyethyleneglycol (PEG) chain; providing an active compound; providingan aqueous solution; combining the composition, compound and solution toform a liposome suspension; and administering the liposome suspensionorally in the form selected from the group comprising a two piece hardgelatin capsule, a soft gelatin capsule, or drops.
 36. A method oforally or sublingually administering a therapeutic compound, the methodcomprising: providing a composition including one or more lipids, wherethe lipids as an aggregate have a P_(a) between about 0.84 and 0.88, aP_(v) between about 0.88 and 0.93 and a melting temperature of betweenabout 0 to 100 degrees centigrade; and where at least one lipid includesa polyethyleneglycol (PEG) chain; providing an active compound;providing an aqueous solution; combining the composition, compound andsolution to form a liposome suspension; and administering the liposomesuspension orally by spraying it into the mouth.
 37. A method ofadministering a therapeutic compound, the method comprising: providing acomposition including one or more lipids, where the lipids as anaggregate have a P_(a) between about 0.84 and 0.88, a P_(v) betweenabout 0.88 and 0.93 and a melting temperature of between about 0 to 100degrees centigrade; and where at least one lipid includes apolyethyleneglycol (PEG) chain; providing an active compound; providingan aqueous solution; combining the composition, compound and solution toform a liposome suspension; incorporating the composition into a formselected from the group comprising a cream, lotion, and gel; andadministering the liposome suspension.